The following abbreviations are used in the present specification.
GAG: glycosaminoglycan
HexN: hexosamine
HexNAc: N-acetylhexosamine
GalN: galactosamine
GalNAc: N-acetylgalactosamine
GlcN: glucosamine
GlcNAc: N-acetylglucosamine
GlcNS: N-sulfoglucosamine
HexA: hexuronic acid
GlcA: glucuronic acid
IdoA: iduronic acid
ΔHexA: unsaturated hex conic acid
CS: chondroitin sulfate
DS: dermatan sulfate
CH: chondroitin
dCH: desulfated chondroitin sulfate
HA: hyaluronic acid
HPN: heparosan
NAH: N-acetylheparosan
HS: heparan sulfate
HEP: heparin
dHEP: desulfated heparin
AS: acharan sulfate
ACH: acharan (2-O-desulfated AS)
KS: keratan sulfate
KPS: keratan polysulfate
ΔDi-0S: ΔHexAα1-3GalNAc
ΔDi-6S: ΔHexAα1-3GalNAc(6S)
ΔDi-4S: ΔHexAα1-3GalNAc(4S)
ΔDi-2S: ΔHexA(2S)α1-3GalNAc
ΔDi-diSD: ΔHexA(2S)α1-3GalNAc(6S)
ΔDi-diSE: ΔHexAα1-3GalNAc(4S,6S)
ΔDi-diSB: ΔHexA(2S)α1-3GalNAc(4S)
ΔDi-triS: ΔHexA(2S)α1-3GalNAc(4S,6S)
ΔDiHA-0S: ΔHexAα1-3GlcNAc
ΔDiHA-6S: ΔHexAα1-3GlcNAc(6S)
ΔDiHA-4S: ΔHexAα1-3GlcNAc(4S)
ΔDiHA-2S: ΔHexA (2S)α1-3GlcNAc
ΔDiHS-0S: ΔHexAα1-4GlcNAc
ΔDiHS-NS: ΔHexAα1-4GlcNS
ΔDiHS-6S: ΔHexAα1-4GlcNAc(65)
ΔDiHS-2S: ΔHexA(2S)α1-4GlcNAc
ΔDiHS-diS1: ΔHexAα1-4Glc(NS, 6S)
ΔDiHS-diS2: ΔHexA(2S)α1-4GlcNS
ΔDiHS-diS3: ΔHexA(2S)α1-4GlcNAc(6S)
ΔDiHS-triS: ΔHexA(2S)α1-4Glc(NS,6S)
In the above description, “α1-3” refers to an α1-3 glycosidic bond, “α1-4” an α1-4 glycosidic bond, “6S” a 6-O-sulfate group, “4S” a 4-O-sulfate group, and “2S” a 2-O-sulfate group, respectively.
Known techniques for sulfating a glycosaminoglycan (GAG) include chemical synthesis, enzymatic synthesis, and a combination thereof (i.e., chemo-enzymatic synthesis). A known method for sulfating a hydroxy group of a GAG (O-sulfation method) through chemical synthesis involves the use of an organic solvent. A known O-sulfation method involves conversion of a GAG into a quaternary ammonium salt form, dissolution of the GAG salt in an organic solvent, and addition of a sulfating agent to the solution for O-sulfation, for example, dissolution of tributylamine salt of N-acetylheparosan in N,N-dimethylformamide, and addition of a sulfur trioxide-pyridine complex to the solution for O-sulfation (Patent Document 1). Another known O-sulfation method involves dissolution of a GAG in a polar organic solvent, and addition of a sulfating agent to the solution for O-sulfation; for example, dissolution of chondroitin in formamide, and addition of a sulfur trioxide-triethylamine complex to the solution for O-sulfation (Patent Document 2). In a method using an organic solvent, the sulfation reaction is terminated by, for example, addition of an aqueous solution containing a salt for pH adjustment (e.g., sodium acetate), since the sulfating agent is inactivated by water (Patent Document 2).
A known method for sulfating an amino group of a GAG (N-sulfation method) through chemical synthesis involves dissolution of an N-deacetylated GAG in a weakly basic aqueous solution having a pH of about 10 (e.g., an aqueous sodium carbonate solution or an aqueous sodium hydrogen carbonate solution), and addition of a sulfating agent to the solution for N-sulfation. Examples of known N-sulfation methods include a method involving N-deacetylation of chondroitin sulfate by hydrazine decomposition, dissolution of the N-deacetylated product in an aqueous sodium carbonate solution, and addition of a sulfur trioxide-triethylamine complex to the solution for N-sulfation; and a method involving N-deacetylation of N-acetylheparosan (K5 polysaccharide) by alkaline hydrolysis, neutralization of the N-deacetylated product, and addition of sodium carbonate and a sulfur trioxide-pyridine complex to the neutralized product for N-sulfation (Non-Patent Documents 1 and 2). However, such a method involving sulfation reaction in a weakly basic aqueous solution specifically sulfates an amino group of a GAG, and fails to sulfate a hydroxy group of a GAG.